Ticks are important ectoparasites and vectors of a variety of pathogens in both animals and humans, and their increasing global distribution poses a growing health risk. Unlike other blood-feeding vectors, ticks feed for an extended period at each life stage and rely exclusively on blood for development and reproduction. Blood digestion in ticks is mediated by a complex multienzyme network within the endolysosomal system of the midgut (MG) epithelial cells. Previous studies have focused largely on protein digestion during the slow feeding phase. However, the processing of the blood meal after the mating-induced rapid engorgement ("big sip") remains unclear, although the rapid turnover of proteins from host blood proteins into yolk proteins in fully fed females is a crucial step for tick reproduction. In this study, we performed a label-free quantitative proteomic analysis of MG tissue extracts and MG contents of the hard tick Ixodes ricinus to characterize proteases and protease inhibitors expressed during selected timepoints of female feeding and off-host digestion. In addition, we analyzed the distribution of digestive enzymes by activity profiling in MG extracts and contents with specific diagnostic substrates. Our results show that the multienzyme network, mainly based on aspartic acid and cysteine cathepsins and complemented by specific types of serine proteases and metalloproteases, is involved in the intracellular and probably also in the luminal digestion of blood meal proteins in fully engorged female ticks. We also detected different types of protease inhibitors and proposed their regulatory role in controlling both endogenous (tick-derived) and host protease activities in the MG tissue and luminal contents storing ingested blood. These results provide comprehensive insights into the physiology of the tick MG and offer new opportunities for the development of future control strategies against ticks and tick-borne diseases.

• Klíčová slova
• adult Ixodes ricinus, label-free proteomics, midgut proteome, proteolytic system, tick physiology,
• MeSH
• klíště * metabolismus   fyziologie   enzymologie   MeSH
• proteasy metabolismus   MeSH
• proteiny členovců * metabolismus   MeSH
• proteom * metabolismus   MeSH
• proteomika * metody   MeSH
• stravovací zvyklosti MeSH
• trávení * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteasy MeSH
• proteiny členovců * MeSH
• proteom * MeSH

Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

• Klíčová slova
• Borrelia, Ixodes, antimicrobial peptides, label-free quantification, lipid metabolism, midgut, protease inhibitors, proteases, proteome, ticks,
• MeSH
• klíště * parazitologie   MeSH
• proteom MeSH
• proteomika MeSH
• trávicí systém MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH

Ticks are blood-feeding arthropods that use the components of their salivary glands to counter the host's hemostatic, inflammatory, and immune responses. The tick midgut also plays a crucial role in hematophagy. It is responsible for managing blood meals (storage and digestion) and protecting against host immunity and pathogen infections. Previous transcriptomic studies revealed the complexity of tick sialomes (salivary gland transcriptomes) and mialomes (midgut transcriptomes) which encode for protease inhibitors, lipocalins (histamine-binding proteins), disintegrins, enzymes, and several other tick-specific proteins. Several studies have demonstrated that mammalian hosts acquire tick resistance against repeated tick bites. Consequently, there is an urgent need to uncover how tick sialomes and mialomes respond to resistant hosts, as they may serve to develop novel tick control strategies and applications. Here, we mimicked natural repeated tick bites in a laboratory setting and analyzed gene expression dynamics in the salivary glands and midguts of adult female ticks. Rabbits were subjected to a primary (feeding on a naive host) and a secondary infestation of the same host (we re-exposed the hosts but to other ticks). We used single salivary glands and midguts dissected from individual siblings adult pathogen-free female Ixodes ricinus to reduce genetic variability between individual ticks. The comprehensive analysis of 88 obtained RNA-seq data sets allows us to provide high-quality annotated sialomes and mialomes from individual ticks. Comparisons between fed/unfed, timepoints, and exposures yielded as many as 3000 putative differentially expressed genes (DEG). Interestingly, when classifying the exposure DEGs by means of a clustering approach we observed that the majority of these genes show increased expression at early feeding time-points in the mid-gut of re-exposed ticks. The existence of clearly defined groups of genes with highly similar responses to re-exposure suggests the existence of molecular swiches. In silico functional analysis shows that these early feeding reexposure response genes form a dense interaction network at protein level being related to virtually all aspects of gene expression regulation and glycosylation. The processed data is available through an easy-to-use database-associated webpage (https://arn.ugr.es/IxoriDB/) that can serve as a valuable resource for tick research.

• Klíčová slova
• midgut, repeated exposure, salivary glands, ticks, transcriptome,
• MeSH
• klíště * genetika   MeSH
• kousnutí klíštětem * MeSH
• králíci MeSH
• obratlovci MeSH
• proteiny členovců genetika   metabolismus   MeSH
• savci genetika   MeSH
• slinné žlázy metabolismus   MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny členovců MeSH

Ticks, notorious blood-feeders and disease-vectors, have lost a part of their genetic complement encoding haem biosynthetic enzymes and are, therefore, dependent on the acquisition and distribution of host haem. Solute carrier protein SLC48A1, aka haem-responsive gene 1 protein (HRG1), has been implicated in haem transport, regulating the availability of intracellular haem. HRG1 transporter has been identified in both free-living and parasitic organisms ranging from unicellular kinetoplastids, nematodes, up to vertebrates. However, an HRG1 homologue in the arthropod lineage has not yet been identified. We have identified a single HRG1 homologue in the midgut transcriptome of the tick Ixodes ricinus, denoted as IrHRG, and have elucidated its role as a haem transporter. Data from haem biosynthesis-deficient yeast growth assays, systemic RNA interference and the evaluation of gallium protoporphyrin IX-mediated toxicity through tick membrane feeding clearly show that IrHRG is the bona fide tetrapyrrole transporter. We argue that during evolution, ticks profited from retaining a functional hrg1 gene in the genome because its protein product facilitates host haem escort from intracellularly digested haemoglobin, rendering haem bioavailable for a haem-dependent network of enzymes.

• Klíčová slova
• HRG, auxotrophy, haem, ticks, transporter,
• MeSH
• fylogeneze MeSH
• hem metabolismus   MeSH
• hemoglobiny metabolismus   MeSH
• hemoproteiny genetika   metabolismus   MeSH
• infestace klíšťaty parazitologie   MeSH
• klíště metabolismus   MeSH
• proteiny členovců genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• transkriptom MeSH
• trávicí systém metabolismus   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hem MeSH
• hemoglobiny MeSH
• hemoproteiny MeSH
• proteiny členovců MeSH

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

• Klíčová slova
• Ixodes ricinus, cathepsin, crystal structure, cysteine protease, digestion, midgut, parasite,
• MeSH
• cystatiny metabolismus   MeSH
• fylogeneze MeSH
• kathepsin L metabolismus   MeSH
• klíšťata metabolismus   MeSH
• klíště metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• trávicí systém metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystatiny MeSH
• kathepsin L MeSH
• krevní proteiny MeSH

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

• Klíčová slova
• Ixodes ricinus, X-ray crystallography, adaptive immunity, blood coagulation, inflammation, saliva, serpin, tick,
• MeSH
• adaptivní imunita účinky léků   MeSH
• aktivace lymfocytů účinky léků   MeSH
• antikoagulancia izolace a purifikace   farmakologie   MeSH
• cytokiny metabolismus   MeSH
• hemokoagulace účinky léků   MeSH
• hmyzí proteiny izolace a purifikace   farmakologie   MeSH
• imunologické faktory izolace a purifikace   farmakologie   MeSH
• inhibitory proteas izolace a purifikace   farmakologie   MeSH
• klíště metabolismus   MeSH
• králíci MeSH
• kultivované buňky MeSH
• lidé MeSH
• lymfocyty účinky léků   imunologie   metabolismus   MeSH
• morčata MeSH
• myši inbrední C3H MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• slezina účinky léků   imunologie   metabolismus   MeSH
• slinné proteiny a peptidy izolace a purifikace   farmakologie   MeSH
• sliny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• morčata MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antikoagulancia MeSH
• cytokiny MeSH
• hmyzí proteiny MeSH
• imunologické faktory MeSH
• inhibitory proteas MeSH
• slinné proteiny a peptidy MeSH

Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.

• MeSH
• arachnida jako vektory mikrobiologie   MeSH
• bakteriální vakcíny aplikace a dávkování   MeSH
• Borrelia burgdorferi komplex účinky léků   MeSH
• infestace klíšťaty genetika   mikrobiologie   prevence a kontrola   přenos   MeSH
• klíště účinky léků   MeSH
• lymeská nemoc genetika   mikrobiologie   prevence a kontrola   přenos   MeSH
• myši MeSH
• proteiny členovců genetika   MeSH
• slinné žlázy mikrobiologie   MeSH
• transkriptom * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální vakcíny MeSH
• proteiny členovců MeSH

During feeding on vertebrate hosts, ticks secrete saliva composed of a rich cocktail of bioactive molecules modulating host immune responses. Although most of the proteinaceous fraction of tick saliva is of little immunogenicity, repeated feeding of ticks on mammalian hosts may lead to impairment of tick feeding, preventing full engorgement. Here, we challenged rabbits with repeated feeding of both Ixodes ricinus nymphs and adults and observed the formation of specific antibodies against several tick salivary proteins. Repeated feeding of both I. ricinus stages led to a gradual decrease in engorged weights. To identify the salivary antigens, isolated immunoglobulins from repeatedly infested rabbits were utilized for a protein pull-down from the saliva of pilocarpine-treated ticks. Eluted antigens were first identified by peptide mass fingerprinting with the aid of available I. ricinus salivary gland transcriptomes originating from early phases of tick feeding. To increase the authenticity of immunogens identified, we also performed, for the first time, de novo assembly of the sialome from I. ricinus females fed for six days, a timepoint used for pilocarpine-salivation. The most dominant I. ricinus salivary immunogens identified in our study were zinc-dependent metalloproteases of three different families. To corroborate the role of metalloproteases at the tick/host interface, we fed ticks micro-injected with a zinc metalloprotease inhibitor, phosphoramidon, on a rabbit. These ticks clearly failed to initiate feeding and to engorge. However, neither feeding to ticks immune blood of repeatedly infested rabbits, nor phosphoramidon injection into ticks, prevented their engorgement when fed in vitro on an artificial membrane system. These data show that Zn metalloproteases play a decisive role in the success of tick feeding, mediated by complex molecular interactions between the host immune, inflammatory, and hemostatic processes, which are absent in in vitro feeding. This basic concept warrants further investigation and reconsideration of the current strategies towards the development of an effective "anti-tick" vaccine.

• Klíčová slova
• Ixodes ricinus, acquired resistance, antigen, immunoprecipitation, metalloprotease, ticks, vaccine,
• MeSH
• infestace klíšťaty * MeSH
• klíště * MeSH
• králíci MeSH
• metaloproteasy MeSH
• proteiny členovců MeSH
• slinné proteiny a peptidy MeSH
• slinné žlázy MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• metaloproteasy MeSH
• proteiny členovců MeSH
• slinné proteiny a peptidy MeSH

BACKGROUND: The castor bean tick Ixodes ricinus is an important vector of several clinically important diseases, whose prevalence increases with accelerating global climate changes. Characterization of a tick life-cycle is thus of great importance. However, researchers mainly focus on specific organs of fed life stages, while early development of this tick species is largely neglected. METHODS: In an attempt to better understand the life-cycle of this widespread arthropod parasite, we sequenced the transcriptomes of four life stages (egg, larva, nymph and adult female), including unfed and partially blood-fed individuals. To enable a more reliable identification of transcripts and their comparison in all five transcriptome libraries, we validated an improved-fit set of five I. ricinus-specific reference genes for internal standard normalization of our transcriptomes. Then, we mapped biological functions to transcripts identified in different life stages (clusters) to elucidate life stage-specific processes. Finally, we drew conclusions from the functional enrichment of these clusters specifically assigned to each transcriptome, also in the context of recently published transcriptomic studies in ticks. RESULTS: We found that reproduction-related transcripts are present in both fed nymphs and fed females, underlining the poorly documented importance of ovaries as moulting regulators in ticks. Additionally, we identified transposase transcripts in tick eggs suggesting elevated transposition during embryogenesis, co-activated with factors driving developmental regulation of gene expression. Our findings also highlight the importance of the regulation of energetic metabolism in tick eggs during embryonic development and glutamate metabolism in nymphs. CONCLUSIONS: Our study presents novel insights into stage-specific transcriptomes of I. ricinus and extends the current knowledge of this medically important pathogen, especially in the early phases of its development.

• Klíčová slova
• Ixodes ricinus, Life stage, Reference gene validation, Tick development, Transcriptome assembly,
• MeSH
• klíště genetika   růst a vývoj   MeSH
• nymfa růst a vývoj   MeSH
• rozmnožování genetika   MeSH
• stadia vývoje MeSH
• stanovení celkové genové exprese * MeSH
• stravovací zvyklosti MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ticks salivate while feeding on their hosts. Saliva helps blood feeding through host anti-hemostatic and immunomodulatory components. Previous transcriptomic and proteomic studies revealed the complexity of tick saliva, comprising hundreds of polypeptides grouped in several multi-genic families such as lipocalins, Kunitz-domain containing peptides, metalloproteases, basic tail secreted proteins, and several other families uniquely found in ticks. These studies also revealed that the composition of saliva changes with time; expression of transcripts from the same family wax and wane as a function of feeding time. Here, we examined whether host immune factors could influence sialome switching by comparing sialomes of ticks fed naturally on a rabbit, to ticks artificially fed on defibrinated blood depleted of immune components. Previous studies were based on transcriptomes derived from pools of several individuals. To get an insight into the uniqueness of tick sialomes, we performed transcriptomic analyses of single salivary glands dissected from individual adult female I. ricinus ticks. Multivariate analysis identified 1,279 contigs differentially expressed as a function of time and/or feeding mode. Cluster analysis of these contigs revealed nine clusters of differentially expressed genes, four of which appeared consistently across several replicates, but five clusters were idiosyncratic, pointing to the uniqueness of sialomes in individual ticks. The disclosure of tick quantum sialomes reveals the unique salivary composition produced by individual ticks as they switch their sialomes throughout the blood meal, a possible mechanism of immune evasion.

• MeSH
• klíště genetika   metabolismus   MeSH
• králíci MeSH
• lidé MeSH
• sekvenční analýza RNA MeSH
• slinné žlázy metabolismus   MeSH
• sliny metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH